We propose to continue our studies on the molecular genetic analysis of two early gene regions of human adenovirus 2 (Ad 2). Our studies will focus on the transforming early region E1a and early gene block Ll and will have three major goals. Firstly, using various mutants, we will identify the functional regions essential for E1a mediated immortalization and transformation of epithelial cells and determine their relationship to induction of cellular DNA synthesis and regulation of the cell division cycle. We will attempt to separate the immortalization and transformation functions controlled by one of the E1a T antigens (243R) and elucidate the possiblemechanisms of immortalization and transformation. Our recent studies have revealed that a domain of the 289R protein suppresses immortalization of primary baby rat kidney cells. The second aspect of our study would be to gain insight into the possible mechanisms of immortalization suppression. We propose to identify the cellular genes differentially regulated by the 289R T antigen or the cellular protein factors that may specifically interact with this protein. If exploratory studies indicate that the 289R protein may be a metalloprotein, the effect of the immortalization up mutations on potential metal binding will be investigated. The third major goal of our study would be to elucidate the functions of the 52- 55kD proteins coded by the L1 region in regulation of viral early, intermediate late and true late gene expression using suppressible nonsense mutants or partially defective deletion and/or insertion mutants.